Introduction

The use of bisphenol A (BPA)-based plastics in food and beverage containers has been a point of controversy for the past decade. Although these clear, tough plastics have been commercially incorporated into the linings of containers such as soup cans, water bottles, and water pipes since the 1950’s, concerns exist about BPA’s xenoestrogenic nature1,2. BPA has been loosely linked to neurological effects, cancer, obesity, and reproductive disorders3.

Uncertainty regarding the potential negative health effects of BPA has led government agencies such as the US Food and Drug Administration (US FDA) and the European Food and Safety Authority (EFSA) to begin a series of ongoing safety studies2. Ultimately, a Tolerable Daily Intake (TDI) of 0.05 mg BPA/kg body weight (b.w.)/day was concluded to be safe. Additionally, the current food migration limit for BPA in the European Union (EU) was set at 0.6 mg/kg, or 600 ppb4.

This study demonstrates the use of a molecularly imprinted polymer (MIP) to selectively extract and concentrate BPA from chicken and beef broth matrices for low-level quantitation. HPLC analysis is subsequently performed using an Ascentis® Express C18 HPLC column.

Experimental

BPA Stock Solution(s)
A 1 mg/mL BPA solution was made by weighing 100 mg of Bisphenol A (Product No. 239658) into a 100 mL volumetric flask and filling the flask to the 100 mL volume with acetonitrile. The BPA was dissolved with stirring for 10 minutes. Another BPA solution (3 µg/mL) was made by combining 15 µL of the 1 mg/mL stock with 4985 µL of acetonitrile:water (40:60) solution. The solution was mixed for 30 seconds using a laboratory vortex mixer.

Calibration Curve
A calibration curve was constructed using dilutions of the 3 µg/mL BPA stock solution. The following concentrations were chosen: 60, 150, 300, 600, and 900 ng/mL. The calibration analysis was carried out under the HPLC conditions listed in Figure 1, and gave a correlation of R2 0.9994. All BPA concentrations for recoveries were calculated by comparison to the calibration curve.

Preparation of Broth Samples
Nationally recognized brands of beef broth (condensed) and canned chicken broth (uncondensed) were purchased for use in the study. A volume of 100 mL of each broth was vacuum filtered through 2.5 µm qualitative filter paper using a ceramic Büchner funnel and side arm Erlenmeyer flask with vacuum. The filtered broth was transferred to glass jars and stored under refrigeration. Broth samples were returned to room temperature prior to use.

Preparation of the Spiked Samples
Samples spiked with BPA (60 ng/mL) were made by combining 19.6 mL aliquots of water or chicken broth with 400 µL of the 3 µg/mL BPA stock solution. After spiking, the samples were shaken at medium speed for 5 minutes on a laboratory shaker. Preliminary tests indicated high levels of BPA were extant in the beef broth. As a result, the beef broth samples were processed without BPA fortification.

Sample Cleanup and Analysis
The SPE cleanup protocol using the SupelMIP SPE-BPA was applied to water, chicken broth, and beef broth samples. During SPE processing, drop rates were easily controlled, and evaporation time was relatively short (15–20 minutes). HPLC analysis was performed on an Ascentis® Express C18 column with florescence detection. The SPE cleanup and analysis procedure is described in the conditions section of Figure 1.

Results and Discussion

Analysis revealed that the unfortified beef broth samples contained an incurred BPA concentration of 119 ng/mL. The chicken broth sample blank contained 8 ng/mL of incurred BPA. Low levels of BPA were also detected in the mobile phase. Spiked samples were corrected for background BPA by subtracting the BPA area response observed in associated blank and mobile phase samples from the average response of spiked samples before final concentrations were calculated.

The average percent recoveries were greater than 72%, with %RSD values below 8% in all cases. Variability was very low in chicken and beef samples.

Beef and chicken samples showed variance in the matrix interference removal. Minor interference was observed to occur in chicken broth at the BPA retention time. The interference was addressed via manual integration. Beef broth samples were interference free at the BPA retention time (Figure 2).

Figure 2. Examination of the Matrix Interferences in the HPLC Analysis of
(a) Chicken Broth Spiked with BPA at 60 ng/mL and
(b) Unfortified Beef Broth Sample with Incurred BPA after SupelMIP SPE-Bisphenol A Cleanup.
Conditions are the same as Figure 1.

Conclusion

This application illustrated the use of the SupelMIP SPE-Bisphenol A for the extraction of BPA from beef and chicken broth matrices prior to chromatographic analysis. In addition to excellent analyte recovery and reproducibility, the MIP SPE extraction and cleanup method provided sufficient matrix interference removal, facilitating quantitation. In addition, this method allowed for detection and quantification of BPA levels that were 10 times lower than those specified by current EU regulations. Therefore, the SupelMIP SPE-Bisphenol A cartridge can be successfully used for quantitation of BPA at a concentration as low as 60 ng/g.

Legal Information

Ascentis and SupelMIP are registered trademarks of Sigma-Aldrich Co. LLC